Novel chimeric rev, tat, and nef antigens

ABSTRACT

This invention provides novel HIV antigens comprising chimeric rev, tat, and nef for use in inducing an immune response. The novel antigens can be used as vaccines to prevent and/or attenuate HIV infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority of U.S. ProvisionalApplication No. 60/332,433, filed Nov. 16, 2001, which is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to improved methods of inducing an immuneresponse for the prevention or treatment of human immunodeficiency virus(HIV) infection by using a novel chimeric antigen that comprisesgenetically modified and re-assorted rev, tat, and nef genes.

BACKGROUND OF THE INVENTION

An effective vaccine for treatment or prevention of HIV infectiondesirably induces a high frequency of CTL responses to multipleepitopes, as virus specific CD8+ T-cell responses have been associatedwith viremia containment in HIV or SIV infected humans or macaques,respectively (Allen, T. M. et al., Nature, 407:386-390 (2000); Borrow etal., J. Virol., 68:6103-6110 (1994); Borrow et al., Nat.Med., 3:205-211(1997); Goulder et al., AIDS, 13 Suppl A:S121-S136 (1999); Koup et al.,J. Virol., 68:4650-4655 (1994); Letvin et al., Science, 280:1875-1880(1998); McMichael et al., Nature, 410:980-987 (2001); Rinaldo et al., J.Virol., 69:5838-5842 (1995); Rowland-Jones et al., Curr Opin Immunol,7:448-455 (1995); Schmitz et al., Science, 283:857-860 (1999)). Most ofthe vaccines developed against HIV-1, SIV-1, or chimeric SHIV viruseshave included the viral structural env and gag genes, and, in somecases, the protease gene. These vaccines, however, have not preventedviral infection, although, in some cases, a milder disease course wasobserved (Amara et al., Science, 292:69-74 (2001); Benson et al., J.Virol., 72:4170-4182 (1998); Hanke et al., J. Virol., 73:7524-7532(1999); Hirsch et al., J. Virol., 70:3741-3752 (1996); Kent et al., J.Virol., 72:10180-10188 (1998); Ourmanov et al., J. Virol., 74:2740-2751(2000); Robinson et al., Nat.Med., 5:526-534 (1999); Seth et al., J.Virol., 74:2502-2509 (2000)).

The response against Rev, Tat, and Nef proteins, which are expressedearly in the virus life cycle (Cullen, FASEB J, 5:2361-2368 (1991);Robert-Guroffet al., J. Virol., 64:3391-3398 (1990)), may beparticularly important in viral containment because their recognitionmay occur before Nef down-modulates MHC class I molecules on the surfaceof the infected cells (Collins et al., Nature, 391:397-401 (1998)) priorto assembly of viral particles. This would therefore provide a window ofopportunity to the immune system to eliminate the infected cell beforevirus is released. In addition, as Nef protects HIV-infected cells fromapoptosis (Antoni et al., J Virol, 69:2384-2392 (1995); Cullen, FASEB J,5:2361-2368 (1991); Robert-Guroff et al., J. Virol., 64:3391-3398(1990); Yoon et al., AIDS Res Hum Retroviruses, 17:99-104 (2001)), aprompt recognition of Nef-expressing cells by cytotoxic T-lymphocytes(CTL) may increase the chances of eliminating virus-infected cells. Thefunctional domains of Tat and Rev proteins are relatively well conservedamong different HIV-1 clades (Meyers et al., B. Korber (ed.), Humanretroviruses and AIDS. Theoretical Biology and Biophysics, Los AlamosNational Laboratory, Los Alamos, p. 55-56 (1995)) and are targets ofCTL's in HIV-1-infected individuals, particularly in the acute phase ofinfection (Addo et al., Proc Natl Acad Sci USA, 98:1781-1786 (2001);Novitsky et al., J Virol, 75:9210-9228 (2001); van Baalen et al., J GenVirol, 78 (Pt 8):1913-1918 (1997)). Responses to these viral proteinshave been identified also in long term non-progressor individuals whocontain viremia in an absence of antiretroviral therapy (Addo et al.,Proc Natl Acad Sci USA, 98:1781-1786 (2001); Froebel et al., AIDS ResHum Retroviruses, 10 Suppl 2:S83-S88 (1994); Novitsky et al., J Virol,75:9210-9228 (2001)). Induction of immune responses to Tat and Rev maytherefore also be important in protection from or potentiation of theimmune response to HIV.

In particular, CTL responses directed against functionally importantdomains of these regulatory proteins may reduce the emergence of viralimmune escape, as mutation at these sites may reduce viral fitness(Nietfield et al., J Immunol, 154:2189-2197 (1995)). Indeed, in somepatients, the relative frequency of CTL recognition has been reported tobe higher for Rev, Tat, and Nef than reverse transcriptase, Env gp41, orgp120 epitopes (Addo et al., Proc Natl Acad Sci USA,98:1781-1786(2001)). The relatively high frequency of anti-Tat responseobserved in some studies could be explained by its availability inextracellular compartments and uptake from cells, as implied by in vitrostudies (Cafaro et al., Nat. Med., 5:643-650 (1999); Kim et al., JImmunol, 159:1666-1668 (1997)).

The ability of early regulatory protein-specific CTL to exertimmunological pressure has been demonstrated in the macaque model inwhich selection for viral immune escape variants in Tat (Allen, T. M. etal., Nature, 407:386-390 (2000)) and Nef (Evans et al., Nat. Med.,5:1270-1276 (1999); Mortara et al., Virology, 278:551-561 (2000)) occursfrequently during primary SIVmac infection. In this model, as well as inhumans infected with HIV-1, the presence of humoral and cellular immuneresponses against Tat (Froebel et al., AIDS Res Hum Retroviruses, 10Suppl 2:S83-S88 (1994); Re et al., J Acquir Immune Defic Syndr HumRetrovirol, 10:408-416 (1995); Rodman et al., Proc Natl Acad Sci USA,90:7719-7723 (1993); Venet et al., J Immunol, 148:2899-2908 (1992);Zagury et al., J Hum Virol, 1:282-292 (1998)) and CTL responses againstRev (van Baalen et al., J Gen Virol, 78 (Pt 8):1913-1918 (1997); vanBaalen et al., J Virol, 72:6851-6857 (1998)) and Nef (Sriwanthana etal., AIDS Res Hum Retroviruses, 17:719-734 (2001)) have beendemonstrated to inversely correlate with disease progression.

The potential benefits of the inclusion of regulatory proteins as partof vaccines against immunodeficiency viruses has been investigated. Inmacaques, immunization with biologically active Tat protein (Cafaro etal., Nat. Med., 5:643-650 (1999)), Tat toxoid (Pauza et al., Proc. Natl.Acad. Sci. USA, 97:3515-3519 (2000)), Tat-encoding DNA vaccine (Cafaroet al., Vaccine, 19:2862-2877 (2001)), or recombinant vaccinia vectorsexpressing Tat and Rev proteins (Osterhaus et al., Vaccine, 17:2713-2714(1999)) did not protect from infection and have demonstrated variouslevels of attenuation of virus replication and disease progression,depending on the animal model used.

A protective effect of Nef-specific CTLs has been established in a studyin macaques, where an inverse correlation was found between thevaccine-induced Nef-specific CTL precursor frequency and virus loadmeasured after challenge (Gallimore et al., Nat. Med., 1:1167-1173(1995)). However, other studies have shown only limited immune responseselicited by vaccination with early regulatory genes and no protectionagainst viral challenge (Calarota et al., J. Immunol., 163:2330-2338(1999); Nilsson et al., Vaccine, 19:3526-3536 (2001); Putkonen et al.,Virology, 250:293-301 (1998)). In most studies, except two (Pauza etal., Proc. Natl. Acad. Sci. USA, 97:3515-3519 (2000)), both Tat and Nefproteins have been used without prior modification.

Expression of HIV and SIVmac genes, including Rev, Tat, and Nef, inmammalian cells has proven to be difficult because of a highly distinctcodon bias for adenine and thymidine at the third codon position (Andreet al., J Virol, 72:1497-1503 (1998); Haas et al., Curr Biol, 6:315-324(1996)), which limits their translation efficiency. Furthermore, severalin vitro studies indicate that both Nef and Tat may have negativeeffects on the host immune response. For example, Nef down-modulatesMHC-I, CD4, and CD28 (Carl et al., J Virol, 75:3657-3665 (2001); Collinset al., Nature, 391:397-401 (1998); Swigut et al., EMBO J, 20:1593-1604(2001)), induces T-cell hyporesponsiveness (Collette et al., J BiolChem, 271:6333-6341 (1996); Collette et al., Eur J Immunol, 26:1788-1793(1996)), and up-regulates Fas ligand on the surface of infected cells(Xu et al., J Exp Med, 186:7-16 (1997)). Wild type Tat also exertsimmunosuppressive functions, through inhibition of both antigen-drivenand nonspecific T-cell proliferation (Collette et al., J Biol Chem,271:6333-6341 (1996); Collette et al., Eur J Immunol, 26:1788-1793(1996); Viscidi et al., Science, 246:1606-1608 (1989); Wrenger et al., JBiol Chem, 272:30283-30288 (1997)), induction of T-cell apoptosis(Goldstein, Nat Med, 2:960-964 (1996)), inhibition of phagocytosis byaccessory cells (Zocchi et al., AIDS, 11:1227-1235 (1997)), inhibitionof IL-2 secretion (Poggi et al., J Biol Chem, 273:7205-7209 (1998)), anddown-regulation of MHC class II complexes (Kanazawa et al., Immunity,12:61-70 (2000)).

Other deleterious effects of these regulatory proteins have also beenreported. Tat transactivates multiple cellular genes, including a numberof cytokines, intercellular adhesion molecules, and chemokines(Rubartelli et al., Immunol. Today, 19:543-545 (1998)) and inducesangiogenesis, possibly contributing to the development ofAIDS-associated tumors (Albini et al., Nat Med, 2:1371-1375 (1996). Tatand Rev proteins induce defects in neuronal differentiation and neuronaldeath (Mabrouk et al., FEBS Lett, 289:13-17 (1991); Nath et al., JVirol, 70:1475-1480 (1996) and Nef promotes neoplastic transformation ofimmortalized neural cells in vitro (Kramer-Hammerle et al., AIDS Res HumRetroviruses, 17:597-602 (2001)).

Thus, there is a need to develop safe and efficacious vaccines thatinclude regulatory HIV proteins. The current invention addresses thisneed and provides a novel chimeric gene comprising genetically modifiedand reassorted rev, tat, and nef genes, e.g. retanef. Such proteins,which are typically expressed in the cytoplasm, are safe and immunogenicvaccines for the prevention and treatment of HIV infection.

SUMMARY OF THE INVENTION

The invention provides expression vectors for the prevention ortreatment of HIV infection. In particular, the invention provides anexpression vector comprising a nucleic acid encoding a chimeric rev,tat, and nef polypeptide. The expression vector is often a viral vector,such as an attenuated pox virus vector, e.g., NYVAC, ALVAC, MVA, orfowlpox. Other viral vectors can also be used, these include adenovirusvectors, adeno-associated virus vector, or Venezuelan equineencephalomyelitis virus vectors.

The chimeric polypeptide expressed by the vector typically comprisesfunctional domains of tat and nef that are disrupted. Furthemore, thechimeric polypeptide expressed by the vector can lack a Rev nuclearlocalization signal, a Rev RNA-binding domain, and a Tat RNA-bindingdomain; and/or can lack an N-terminal Nef myristylation signal. In oneembodiment, the expression vector comprises a retanef gene, whichencodes the polypeptide Retanef. Often, the retanef gene is expressedusing a NYVAC or ALVAC vector.

In another aspect, the invention provides a method of inducing an immuneresponse comprising administering a first expression vector comprising anucleic acid sequence encoding a chimeric rev, tat, and nef polypeptide,wherein the expression vector enters the cells of the recipient andintracellularly produces rev, tat, and nef-specific peptides that arepresented on the cell's MHC class I molecules in an amount sufficient tostimulate a CD8⁺ response. The method often employs an expressionvector, e.g., a viral expression vector, encoding retanef.

The method often further comprise administering a second expressionvector comprising a nucleic acid sequence encoding a chimeric rev, tat,and nef polypeptide, e.g., retanef, wherein the second expression vectoris administered as naked DNA. In some embodiments, the naked DNAexpressing the chimeric rev, tat, and nef polypeptide is administeredprior to a viral vector that expressed a chimeric rev, tat, and nefpolypeptide. In other embodiments, the method comprises additional stepsof administering one or more expression vectors, e.g., NYVAC and/ornaked DNA, encoding HIV structural polypeptides such as one or moreepitopes from the gag, pol, and env genes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the chimeric Retanef protein.The exons of Tat, Nef, and Rev are arranged in the configurationdepicted in the figure. C=carboxy terminus. N=amino terminus. The“number” refers to the amino acid number of the proteins according tothe Los Alamos database for SIV.

FIG. 2 shows the nucleotide and amino acid sequence of the chimericretanef gene and the amino acid sequence of the Retanef protein.

FIG. 3 shows tat-specific T-cell proliferation in macaques inoculatedwith DNA/NYVACretanef alone and macaques inoculated withDNA/NYVACretanef and DNA/NYVACgag-pol-env (lower panel).

FIG. 4 shows nef-specific T-cell proliferation in macaques inoculatedwith DNA/NYVACretanef alone and macaques inoculated withDNA/NYVACretanef and DNA/NYVACgag-pol-env (lower panel).

FIG. 5 shows Tat28 tetramer staining in macaques inoculated withDNA/NYVACretanef alone and macaques inoculated with DNA/NYVACretanef andDNA/NYVACgag-pol-env (lower panel).

FIG. 6 shows the level of SIV in the blood of animals vaccinated withthe constructs as indicated for each group.

FIG. 7 shows the statistical significance of the data shown in FIG. 6.

Definitions

“Attenuated recombinant virus” refers to a virus that has beengenetically altered by modern molecular biological methods, e.g.restriction endonuclease and ligase treatment, and rendered lessvirulent than wild type, typically by deletion of specific genes or byserial passage in a non-natural host cell line or at cold temperatures.

A “chimeric rev, tat, and nef polypeptide” refers to a fusion protein,i.e., the sequences are covalently linked, comprising rev, tat, and nefpolypeptide sequences, or subsequences (also referred to as “fragments”or “domains”), thereof. Rev, tat, and nef polypeptide sequences can bein any order in the chimeric molecules. Fragments of the polypeptidescomprising the chimeric constructs can also be interspersed, e.g.,subsequences of rev can be interspersed with subsequences of tat or nef.“Retanef” refers to the construct shown in FIG. 1. The nucleic acid andamino acid sequences of the Retanef construct in FIG. 1 is provided inFIG. 2. The term “Retanef” includes conservatively modified variantsthat induce at least 70%, preferably, 80%, 85%, 90%, or 95% of theimmune response induced by the Retanef polypeptide having the amino acidsequence set forth in FIG. 2. The characterization of such proteins isdescribed in the sections herein describing chimeric rev-tat-nefpolypeptides and methods of analyzing the immune response.

“Efficient CD8⁺ response” is referred to as the ability of cytotoxicCD8⁺ T-cells to recognize and kill cells expressing foreign peptides inthe context of a major histocompatibility complex (MHC) class Imolecule.

“Nonstructural viral proteins” are those proteins that are needed forviral production but are not found as components of the viral particle.They include DNA binding proteins and various enzymes that are encodedby viral genes. The term “proteins” includes both the intact proteinsand fragments of the proteins or peptides which are recognized by theimmune cell as epitopes of the native protein.

A “nucleic acid vaccine” or “naked DNA vaccine” refers to a vaccine thatincludes one or more expression vectors that encodes B-cell and/orT-cell epitopes and provides an immunoprotective response in the personbeing vaccinated. As used herein, the term does not include a viralvaccine, i.e., a vaccine in which the nucleic acid is within a viralcapsid. An “expression vector” refers to any expression vector, e.g.,viral or plasmid.

“Nucleic acid-based vaccines” can include both naked DNA and vectoredDNA within a viral capsid where the nucleic acid encodes B-cell andT-cell epitopes and provides an immunoprotective response in the personbeing vaccinated.

The term “reassorted” as used herein refers to splitting at least one ofrev, tat, or nef proteins into segments that are separated in thechimeric polypeptide such that the activity of the polypeptide or adomain of the polypeptide is disrupted.

“Pox viruses” are large, enveloped viruses with double-stranded DNA thatis covalently closed at the ends. Pox viruses replicate entirely in thecytoplasm, establishing discrete centers of viral synthesis. Their useas vaccines has been known since the early 1980's (see, e.g. Panicali,D. et al. “Construction of live vaccines by using genetically engineeredpox viruses: biological activity of recombinant vaccinia virusexpressing influenza virus hemagglutinin”, Proc. Natl. Acad. Sci. USA80:5364-5368, 1983).

“Potentiating” or “enhancing” an immune response means increasing themagnitude and/or the breadth of the immune response, i.e., the number ofcells induced by a particular epitope may be increased and/or thenumbers of epitopes that are recognized may be increased (“breadth”). A5-fold, often 10-fold or greater, enhancement in both CD8⁺ and CD4⁺T-cell responses is obtained with administration of a combination ofnucleic acid/recombinant virus vaccines compared to administration ofeither vaccine alone.

A “retrovirus” is a virus containing an RNA genome and an enzyme,reverse transcriptase, which is an RNA-dependent DNA polymerase thatuses an RNA molecule as a template for the synthesis of a complementaryDNA strand. The DNA form of a retrovirus commonly integrates into thehost-cell chromosomes and remains part of the host cell genome for therest of the cell's life.

“Viral load” is the amount of virus present in the blood of a patient.Viral load is also referred to as viral titer or viremia. Viral load canbe measured in variety of standard ways. In preferred embodiments, theDNA/recombinant virus prime boost protocol of the invention controlsviremia and leads to a greater reduction in viral load than thatobtained when either vaccine is used alone.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95%identity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Preferably, the identity exists over a region that is at leastabout 20 amino acids or nucleotides in length, or more preferably over aregion that is 25, 35, or 50-100 amino acids or nucleotides in length.

DETAILED DESCRIPTION

Introduction

The human immunodeficiency virus (HIV), regulatory proteins Rev, Tat,and Nef are expressed early after infection and represent attractivetargets to be included in a vaccine for AIDS. However, the low levelexpression of these proteins in mammalian cells and the potentialimmunosuppressive activity of Tat and Nef, as such, represent alimitation to their inclusion in preventive or therapeutic vaccines. Tocircumvent these issues, the current invention provides novelpolynucleotides and polypeptides comprising tat, rev, and nef chimericmolecules. The chimeric molecules of the invention comprises geneticallymodified and re-assorted rev, tat, and nef genes, e.g., retanef, whichencodes an immunogenic protein of approximately 55 kDa. Retanef andother chimeric polypeptides of the invention can be provided as avaccine for the prevention or attentuation of HIV infection.

Typically, the vaccine is a nucleic acid-based vaccine, often a plasmideucaryotic expression vector or a recombinant viral vector, e.g., anattenuated pox viruses vector. The vaccine can be administered toindividuals at risk for infection or individuals who may already beinfected.

Vaccines of Use in this Invention

Vaccines useful for the induction of CD8⁺ T-cell responses comprisenucleic acid-based vaccines (preferably delivered as a DNA-basedvaccine) including naked DNA and viral vectors, e.g., recombinant poxvirus vaccines, that provide for the intracellular production ofviral-specific peptide epitopes that are presented on MHC Class Imolecules and subsequently induce an immunoprotective cytotoxic Tlymphocyte (CTL) response.

The invention typically contemplates single or multiple administrationsof the nucleic acid vaccine in combination with one or moreadministrations of the recombinant virus vaccine. This vaccinationregimen may be complemented with administration of recombinant proteinvaccines, or may be used with additional vaccine vehicles. Preferably,administration of the nucleic acid vaccine precedes administration ofthe recombinant virus vaccine.

In preferred embodiments, the DNA/recombinant virus prime boost protocolcontrols viremia and reduces viral load as well as potentiating a CD8⁺response.

Chimeric rev-tat-nef

Chimeric nucleic acids of the invention comprise the non-structuralgenes rev, tat, and nef of HIV. The polypeptide encoded by the nucleicacid is thus a fusion polypeptide comprising regions of rev, tat, andnef, i.e., the regions of rev, tat, and nef are covalently bonded to oneanother.

The rev, tat, and nef sequences can be from any HIV sequence, includingboth HIV-1 and HIV-2. Rev, tat, and nef nucleic acid and proteinsequences for use in generating the chimeric molecules of the inventionare known, e.g., the Los Alamos database. The HIV-1 and HIV-2 genomes,and the DNA sequences of HIV-1 and HIV-2, and respective strains arealso described in the publication, HIV Sequence Compendium 2000, Kuikenet al, Eds. Theoretical Biology and Biophysics Group, Los AlamosNational Laboratory, Los Almos, N. Mex. Any rev, tat, or nef sequencecan be used to construct the vaccines of the invention.

Construction of the vaccine constructs uses routine techniques in thefield of recombinant genetics. Basic texts disclosing the generalmethods of use in this invention include Sambrook and Russell, MolecularCloning, A Laboratory Manual (3rd ed. 2001, Cold Spring HarborLaboratory Press); Kriegler, Gene Transfer and Expression: A LaboratoryManual (1990); and Current Protocols in Molecular Biology (Ausubel etal., eds., 1994)).

The rev, tat, and nef sequences included in the chimeric proteins of theinvention are typically disrupted and/or altered to inactivate specificfunctions of the individual proteins. The specific functions can beinactivated using a variety of approaches, for example, by shuffling thecoding regions of the nucleic acid sequences encoding the proteins; bydeleting specific regions of the individual proteins, e.g., one or moreamino acid residues in a functional domain of the protein; or bymutagenizing specific sequences to prevent function. Such changes aretypically accomplished so as to prevent disruption of known T-cell, orB-cell, epitopes. Accordingly, these chimeric constructs retainimmunogenicity.

There are many ways of generating alterations in a given nucleic acidsequence. Many methods, for example used amplifcation, e.g., PCRamplifcation strategies, to amplify sequences containing the desiredchanges. Alternatively, a nucleic acid of the invention can bechemically synthesized.

For chemical synthesis, the peptides can be synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. (See, for example, Stewart & Young,SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984).Further, individual fragments of rev, tat, and nef can be joined usingchemical ligation to produce larger peptides that are still within thebounds of the invention.

In addition, nonclassical amino acids or chemical amino acid analogs canbe introduced as a substitution or addition into the sequence.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

Alternatively, recombinant DNA technology can be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, e.g., asdescribed generally in Sambrook and Russell, supra.

Typically, a nucleic acid sequence encoding the chimeric polypeptide isproduced by chemical synthesis or is produced by ligating appropriatefragments to one another. The fragments can be produced chemically, orcan be obtained by PCR or isolated from plasmids or other vectors thatcontain the sequence of interest. The coding sequence can then beprovided with appropriate linkers and ligated into expression vectors(e.g., for vaccines and/or for production of reocmbinant protein)commonly available in the art, and the vectors used to transformsuitable hosts to produce the desired fusion protein. A number of suchvectors and suitable host systems are available. For expression of thefusion proteins, the coding sequence will be provided with operablylinked start and stop codons, promoter and terminator regions andusually a replication system to provide an expression vector forexpression in the desired cellular host. For example, for production ofrecombinant protein, promoter sequences compatible with bacterial hostsare provided in plasmids containing convenient restriction sites forinsertion of the desired coding sequence. The resulting expressionvectors are transformed into suitable bacterial hosts. Of course, yeast,insect or mammalian cell hosts may also be used, employing suitablevectors and control sequences (see, e.g., Sambrook and Russell, supra).

Many conservative variations of the nucleic acid sequences can be usedto generate an essentially identical polypeptide. For example, due tothe degeneracy of the genetic code, “silent substitutions” (i.e.,substitutions of a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Often, the sequencesused in constructing a chimeric gene of the invention employsubstitutions based on codon usage frequencies. For example, the codonusage typical of lentiviruses, which is biased for adenine and thymidineat the third codon position, reduces the translation efficiency inmammalian cells (Andre et al., J Virol, 72:1497-1503 (1998); Haas etal., Curr Biol, 6:315-324 (1996)). Thus, appropriate codons to optimizemammalian expression can be substituted for the lentivirus codons.

Modifications to rev, tat, and nef nucleic acid and polypeptidesequences incorporated into the chimeric molecules, particularly thosewhich result in a change the amino acid sequence, are evaluated byroutine screening techniques to ensure that the chimeric proteins retainimmunogenicity. For instance, the ability of the peptide to induce aCD8+ response can be determined using methodology such as cytotoxic Tcell assays or direct quantification of antigen-specific T cells bystaining with Fluorescein-labeled HLA tetrameric complexes (Altman, J.D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. etal., Science 274:94, 1996). Other assays include staining forintracellular lymphokines, and γ-interferon release assays or ELISPOTassays. Similarly, the ability of the peptide to stimulate CD4+ cellscan also be determined, e.g., by T-cell proliferation assays.

Typically, suitable modified rev, tat, and nef polypeptides, orfragments of the polypeptides, that can be included as components of thechimeric molecules have about 80% amino acid sequence identity,optionally about 75%, 80%, 85%, 90%, or 95-98% amino acid sequenceidentity to a known rev, tat, or nef protein sequence over a comparisonwindow of about 20 amino acids, optionally about 25, 30, or, 50-100amino acids, or the length of the entire protein. The sequence can becompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. For purposes of this patent, percent amino acid identity isdetermined by the default parameters of BLAST.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The comparison window includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 20 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Cross-Reactive Binding to Antibodies

Rev, tat, nef chimeric polypeptides can also be identified by theabilitiy to cross-react with antibodies, preferably polyclonalantibodies, that bind to known rev, tat, and nef, polypeptides. A rev,tat, nef chimeric polypeptide can be tested for cross-reactivity as achimeric molecule or can be tested as using specific fragments of thechimeric molecule that correspond to the rev, tat, or nef domains of thefusion proteins.

Polyclonal antibodies are generated using methods well known to those ofordinary skill in the art (see, e.g., Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual(1988)). Those rev, tat, nef proteins that are immunologicallycross-reactive binding proteins can then be detected by a variety ofassay methods. For descriptions of various formats and conditions thatcan be used, see, e.g., Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993), Coligan, supra, and Harlow & Lane,supra.

Useful immunoassay formats include assays where a sample protein isimmobilized to a solid support. For example, a cross-reactive rev, tat,nef fusion protein can be identified using an immunoblot analysis suchas a western blot. The western blot technique generally compriseselectrophoresing a sample comprising a rev-tat-nef chimeric polypeptideon a gel, transferring the separated proteins to a suitable solidsupport, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with antibodiesthat bind to known rev, tat, or nef polypeptides. The antibodies thenspecifically bind to cross-reactive rev, tat, or nef polypeptides on thesolid support. The antibodies may be directly labeled or alternativelymay be subsequently detected using labeled antibodies (e.g., labeledsheep anti-mouse antibodies) that specifically bind to the rev, tat, ornef antibodies. Other immunoblot assays, such as dot blots, are alsouseful for identifying rev, tat, and nef chimeric molecules suitable foruse in the invention.

Using this methodology under designated immunoassay conditions,immunologically cross-reactive rev, tat, and nef sequences in thechimeric fusion proteins that bind to a particular rev, tat, or nefantibody at least two times the background or more, typically more than10 times background, and do not substantially bind in a significantamount to other proteins present in the sample can be identified.

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, polyclonal antisera thathave been generated to a known, rev, tat, or nef polypeptide, e.g.,HIV-1 rev, tat, or nef can be used. The antisera can be immobilized to asolid support. The ability of added rev, tat, or nef chimeric proteins(or fragments of the rev, tat, or nef chimeric proteins) to compete forbinding with known rev, tat, or nef polypeptides is analyzed bycomparing the binding to a standard curve generated using the knownpolypeptide. The crossreactivity for the proteins is calculated, usingstandard calculations.

In order to make this comparison, the standard protein and test proteinare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding is determined. Ifthe amount of the test rev, tat, and nef chimeric protein, or fragmentof the chimeric protein, required to inhibit 50% of binding is less than10 times the amount of the standard protein that is required to inhibit50% of binding, then test rev, tat, nef protein is said to specificallybind to the polyclonal antibodies generated to the known rev, tat, ornef immunogen.

Rev, tat, and nef Sequences

The rev sequences included in the chimeric polynucleotides andpolypeptides can be modified by deleting specific regions, for example,the nuclear localization sequence (NLS) and/or RNA binding domain ofRev. Deletion of these regions thereby prevents nuclear localization ofa chimeric polypeptide containing rev and, when the RNA binding domainis deleted, prevents binding to its recognition sequence. Functionaldisruption can also be achieved by deleting and/or altering specificamino acids within the functional domains.

Tat sequences can be similarly altered to prevent function. For example,tat can be engineered to delete its nuclear localization sequence and/orRNA binding domain. In many embodiments, the RNA binding domains and NLSregions of both rev and tat are deleted or disrupted to prevent suchactivities in a chimeric peptide of the invention.

Nef includes a myristylation site that is required for translocation ofNef to the cellular membrane and downregulation of the CD4+ and MHC-Imolecules. This sequence is an important determination of viralpathogenicity. The myristylation sequence can be deleted or mutagenizedto prevent translocation of a chimeric protein comprising nef to thecellular membrane.

The ret, nef, and tat nucleic acid sequences can be included in theconstruct in any order. The protein-encoding regions can also bedispersed. For example, one or more of the proteins can be divided intoan N-terminal part and a C-terminal part. These two parts can then beseparated by intervening regions of the other proteins. The fragments orregions of the rev, tat, and nef polypeptide sequences included in thechimeric constructs can vary in size from the full-length polypeptide tofragments of the full-length polypeptide sequences. For example, rev,tat, or nef fragments of about 20, 25, 50, 75, 100, 125, 150, 175, or200, or 250 amino acids in length can be incorporated into the chimericpolypeptides.

Attenuated Recombinant Viral Vaccines

Attenuated recombinant poxviruses that express retrovirus-specificepitopes are typically used in this invention. Attenuated viruses aremodified from their wildtype virulent form to be either symptomless orweakened when infecting humans. Typically, the genome of the virus isdefective in respect of a gene essential for the efficient production oressential for the production of infectious virus. The mutant virus actsas a vector for an immunogenic retroviral protein by virtue of the virusencoding foreign DNA. This provokes or stimulates a cell-mediated CD8⁺response.

The virus is then introduced into a human vaccinee by standard methodsfor vaccination of live vaccines. A live vaccine of the invention can beadministered at, for example, about 10⁴-10⁸ organisms/dose, or 10⁶ to10⁹ pfu per dose. Actual dosages of such a vaccine can be readilydetermined by one of ordinary skill in the field of vaccine technology.

The selection of the virus is not critical. Examples of viral expressionvectors include adenoviruses as described in M. Eloit et al,“Construction of a Defective Adenovirus Vector Expressing thePseudorabies Virus Glycoprotein gp50 and its Use as a Live Vaccine”, J.Gen. Virol., 71(10):2425-2431 (October, 1990).), adeno-associatedviruses (see, e.g., Samulski et al., J. Virol. 61:3096-3101 (1987);Samulski et al., J. Virol. 63:3822-3828 (1989)), papillomavirus, EpsteinBarr virus (EBV) and Rhinoviruses (see, e.g., U.S. Pat. No. 5,714,374).Human parainfluenza viruses are also reported to be useful, especiallyJS CP45 HPIV-3 strain. The viral vector may be derived from herpessimplex virus (HSV) in which, for example, the gene encodingglycoprotein H (gH) has been inactivated or deleted. Other suitableviral vectors include retroviruses (see, e.g., Miller, Human Gene Ther.1:5-14 (1990); Ausubel et al., Current Protocols in Molecular Biology).Other viral expression vectors that can be used as vaccines includealphaviruses, such as Venezuelan equine encephalomyelitis virus (see,e.g, U.S. Pat. Nos. 5,643,576 and 6,296,854).

The poxviruses are often used in this invention. There are a variety ofattenuated poxviruses that are available for use as a vaccine againstHIV. These include attenuated vaccinia virus, cowpox virus and canarypoxvirus. In brief, the basic technique of inserting foreign genes intolive infectious poxvirus involves a recombination between pox DNAsequences flanking a foreign genetic element in a donor plasmid and ahomologous sequences present in the rescuing poxvirus as described inPiccini et al., Methods in Enzymology 153, 545-563 (1987). Morespecifically, the recombinant poxviruses are constructed in two stepsknown in the art and analogous to the methods for creating syntheticrecombinants of poxviruses such as the vaccinia virus and avipox virusdescribed in U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587,and 5,174,993, the disclosures of which are incorporated herein byreference.

First, the DNA gene sequence encoding an antigenic sequence such as aknown T-cell epitope is selected to be inserted into the virus and isplaced into an E. coli plasmid construct into which DNA homologous to asection of DNA of the poxvirus has been inserted. Separately, the DNAgene sequence to be inserted is ligated to a promoter. The promoter-genelinkage is positioned in the plasmid construct so that the promoter-genelinkage is flanked on both ends by DNA homologous to a DNA sequenceflanking a region of pox DNA containing a nonessential locus. Theresulting plasmid construct is then amplified by growth within E. colibacteria.

Second, the isolated plasmid containing the DNA gene sequence to beinserted is transfected into a cell culture, e.g. chick embryofibroblasts, along with the poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively gives apoxvirus modified by the presence, in a nonessential region of itsgenome, of foreign DNA sequences.

Attenuated recombinant pox viruses are a preferred vaccine. A detailedreview of this technology is found in U.S. Pat. No. 5,863,542, which isincorporated by reference herein. These viruses are modified recombinantviruses having inactivated virus-encoded genetic functions so that therecombinant virus has attenuated virulence and enhanced safety. Thefunctions can be non-essential, or associated with virulence. Thepoxvirus is generally a vaccinia virus or an avipox virus, such asfowlpox virus and canarypox virus. The viruses are generated using thegeneral strategy outlined above and in U.S. Pat. No. 5,863,542.

Representative examples of recombinant pox viruses include ALVAC,TROVAC, NYVAC, and vCP205 (ALVAC-MN120TMG). These viruses were depositedunder the terms of the Budapest Treaty with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852, USA:NYVAC under ATCC accession number VR-2559 on Mar. 6, 1997; vCP205(ALVAC-MN120TMG) under ATCC accession number VR-2557 on Mar. 6, 1997;TROVAC under ATCC accession number VR-2553 on Feb. 6, 1997 and, ALVACunder ATCC accession number VR-2547 on Nov. 14, 1996.

NYVAC is a genetically engineered vaccinia virus strain generated by thespecific deletion of eighteen open reading frames encoding gene productsassociated with virulence and host range. NYVAC is highly attenuated bya number of criteria including: i) decreased virulence afterintracerebral inoculation in newborn mice, ii) inocuity in genetically(nu⁺/nu⁺) or chemically (cyclophosphamide) immunocompromised mice, iii)failure to cause disseminated infection in immunocompromised mice, iv)lack of significant induration and ulceration on rabbit skin, v) rapidclearance from the site of inoculation, and vi) greatly reducedreplication competency on a number of tissue culture cell linesincluding those of human origin.

TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolatederived from the FP-1 vaccine strain of fowlpox virus which is licensedfor vaccination of 1 day old chicks.

ALVAC is an attenuated canarypox virus-based vector that was aplaque-cloned derivative of the licensed canarypox vaccine, Kanapox(Tartaglia et al., 1992). ALVAC has some general properties which arethe same as some general properties of Kanapox. ALVAC-based recombinantviruses expressing extrinsic immunogens have also been demonstratedefficacious as vaccine vectors. This avipox vector is restricted toavian species for productive replication. On human cell cultures,canarypox virus replication is aborted early in the viral replicationcycle prior to viral DNA synthesis. Nevertheless, when engineered toexpress extrinsic immunogens, authentic expression and processing isobserved in vitro in mammalian cells and inoculation into numerousmammalian species induces antibody and cellular immune responses to theextrinsic immunogen and provides protection against challenge with thecognate pathogen.

NYVAC, ALVAC and TROVAC have also been recognized as unique among allpoxviruses in that the National Institutes of Health (“NIH”)(U.S. PublicHealth Service), Recombinant DNA Advisory Committee, which issuesguidelines for the physical containment of genetic material such asviruses and vectors, i.e., guidelines for safety procedures for the useof such viruses and vectors which are based upon the pathogenicity ofthe particular virus or vector, granted a reduction in physicalcontainment level: from BSL2 to BSL1. No other poxvirus has a BSL1physical containment level. Even the Copenhagen strain of vacciniavirus-the common smallpox vaccine-has a higher physical containmentlevel; namely, BSL2. Accordingly, the art has recognized that NYVAC,ALVAC and TROVAC have a lower pathogenicity than any other poxvirus.

Another attenuated poxvirus of preferred use for this invention isModified Vaccinia virus Ankara (MVA), which acquired defects in itsreplication ability in humans as well as most mammalian cells followingover 500 serial passages in chicken fibroblasts (see, e.g., Mayr et al.,Infection 3:6-14 (1975); Carrol, M. and Moss, B. Virology 238:198-211(1997)). MVA retains its original immunogenicity and itsvariola-protective effect and no longer has any virulence andcontagiousness for animals and humans. As in the case of NYVAC or ALVAC,expression of recombinant protein occurs during an abortive infection ofhuman cells, thus providing a safe, yet effective, delivery system forforeign antigens.

Nucleic Acid Vaccines

Nucleic acid vaccines, preferably DNA vaccines may also be used in theinvention. Preferably, the nucleic acid vaccines is administered in aregimen that also comprises administration of a viral vaccine. Nucleicacid vaccines as defined herein, typically plasmid expression vectorsthat are not encapsidated in a viral particle. The nucleic acid vaccineis directly introduced into the cells of the individual receiving thevaccine regimen. This approach is described, for instance, in Wolff et.al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859;5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.Examples of DNA-based delivery technologies include, “naked DNA”,facilitated (bupivicaine, polymers, peptide-mediated) delivery, andcationic lipid complexes or liposomes. The nucleic acids can beadministered using ballistic delivery as described, for instance, inU.S. Pat. No. 5,204,253 or pressure (see, e.g., U.S. Pat. No.5,922,687). Using this technique, particles comprised solely of DNA areadministered, or in an alternative embodiment, the DNA can be adhered toparticles, such as gold particles, for administration.

As is well known in the art, a large number of factors can influence theefficiency of expression of antigen genes and/or the immunogenicity ofDNA vaccines. Examples of such factors include the reproducibility ofinoculation, construction of the plasmid vector, choice of the promoterused to drive antigen gene expression and stability of the inserted genein the plasmid.

Any of the conventional vectors used for expression in eukaryotic cellsmay be used for directly introducing DNA into tissue. Expression vectorscontaining regulatory elements from eukaryotic viruses are typicallyused in eukaryotic expression vectors, e.g., SV40 CMB vectors. Otherexemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, and any other vector allowing expression of proteins underthe direction of such promoters as the SV40 early promoter, SV40 laterpromoter, metallothionein promoter, human cytomegalovirus promoter,murine mammary tumor virus promoter, Rous sarcoma virus promoter,polyhedrin promoter, or other promoters shown effective for expressionin eukaryotic cells.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins. If required,supercoiled DNA can be isolated from the open circular and linear formsusing gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is particularly suitable for intramuscular (IM) or intradermal(ID) administration.

To maximize the immunotherapeutic effects of minigene DNA vaccines,alternative methods for formulating purified plasmid DNA may bedesirable. A variety of methods have been described, and new techniquesmay become available. Cationic lipids can also be used in theformulation (see, e.g., as described by WO 93/24640; Mannino &Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833;WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413(1987). In addition, glycolipids, fusogenic liposomes, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Polypeptide Vaccines

The chimeric rev, tat, and nef polypeptides of the invention can also beadministered as vaccines. Theses polypeptides can be produced bychemical synthesis or by recombinant DNA technology as described above.

The chimeric rev, tat, and nef contstructs can also be administered aspeptide. The peptide can be administered with a variety of agents, e.g.,a carrier.

There are a number of strategies for amplifying an immunogen'seffectiveness, particularly as related to the art of vaccines. Theseinclude strategies whereby an immunogenic peptide may be directlymodified to enhance immunogenicity or physical properties such asstability. For example, cyclization or circularization of a peptide canincrease the peptide's antigenic and immunogenic potency. See, e.g.,U.S. Pat. No. 5,001,049 which is incorporated by reference herein.

The immunogenicity of the chimeric rev, tat, and nef proteins may alsobe modulated by coupling to fatty acid moieties to produce lipidatedpeptides. Convenient fatty acid moieties include glycolipid analogs,N-palmityl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteinyl-serine (PAM3Cys-Ser), N-palmityl-S-[2,3 bis (paInitoyloxy)-(2RS)-propyl-[R]-cysteine(TPC), tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS), oradipalmityl-lysine moiety

The polypeptides may also be conjugated to a lipidated amino acid, suchas an octadecyl ester of an aromatic acid, such as tyrosine, includingactadecyl-tryrosine (OTH).

Carriers may also be used with the polypeptide vaccines. Carriers areewell known in the art, and include, e.g., thyroglobulin, albumins suchas human serum albumin, tetanus toxoid, polyamino acids such as polyL-lysine, poly L-glutamic acid, i and the like. The vaccines can containa physiologically tolerable (i.e., acceptable) diluent such as water, orsaline, preferably phosphate buffered saline. The vaccines alsotypically include an adjuvant. Adjuvants such as incomplete Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, or alum are examplesof materials well known in the art.

Characterization of the Immune Response in Vaccinated Individuals

The vaccine regimen can be delivered to individuals at risk forinfection with HIV or to patients who are infected with the virus. Inorder to assess the efficacy of the vaccine, the immune response can beassessed by measuring the induction of CD4⁺, CD8⁺, and antibodyresponses to particular epitopes. Moreover, viral titer can be measuredin patients treated with the vaccine who are already infected. Theseparameters can be measured using techniques well known to those of skillin the art. Examples of such techniques are described below.

CD4⁺ T Cell Counts

To assess the effectiveness of the vaccine combination in a recipientand to monitor the immune system of a patient already infected with thevirus who is a candidate for treatment with the vaccine regimen, it isimportant to measure CD4⁺ T cell counts. A detailed description of thisprocedure was published by Janet K. A. Nicholson, Ph.D et al. 1997Revised Guidelines for Performing CD4+ T-Cell Determinations in PersonsInfected with Human Immunodeficiency Virus (HIV) in The Morbidity andMortality Weekly Report, 46(RR-2):[inclusive page numbers], Feb. 14,1997. Centers for Disease Control.

In brief, most laboratories measure absolute CD4⁺ T-cell levels in wholeblood by a multi-platform, three-stage process. The CD4⁺ T-cell numberis the product of three laboratory techniques: the white blood cell(WBC) count; the percentage of WBCs that are lymphocytes (differential);and the percentage of lymphocytes that are CD4⁺ T-cells. The last stagein the process of measuring the percentage of CD4⁺ T-lymphocytes in thewhole-blood sample is referred to as “immunophenotyping by flowcytometry.

Immunophenotyping refers to the detection of antigenic determinants(which are unique to particular cell types) on the surface of WBCs usingantigen-specific monoclonal antibodies that have been labeled with afluorescent dye or fluorochrome (e.g., phycoerythrin [PE] or fluoresceinisothiocyanate [FITC]). The fluorochrome-labeled cells are analyzed byusing a flow cytometer, which categorizes individual cells according tosize, granularity, fluorochrome, and intensity of fluorescence. Size andgranularity, detected by light scattering, characterize the types ofWBCs (i.e., granulocytes, monocytes, and lymphocytes).Fluorochrome-labeled antibodies distinguish populations andsubpopulations of WBCs.

Systems for measuring CD4⁺ cells are commercially available. For exampleBecton Dickenson's FACSCount System automatically measure absolutesCD4⁺, CD8⁺, and CD3⁺ 0 T lymphocytes. It is a self-contained system,incorporating instrument, reagents, and controls.

A successful increase of CD4⁺ cell counts would be a 2× or higherincrease in the number of CD4⁺ cells.

Measurements of CD8⁺ Responses

CD8⁺ T-cell responses may be measured, for example, by using tetramerstaining of fresh or cultured PBMC, ELISPOT assays or by usingfunctional cytotoxicity assays, which are well-known to those of skillin the art. For example, a functional cytotoxicity assay can beperformed as follows. Briefly, peripheral blood lymphocytes frompatients are cultured with HIV peptide epitope at a density of aboutfive million cells/ml. Following three days of culture, the medium issupplemented with human IL-2 at 20 units/ml and the cultures aremaintained for four additional days. PBLs are centrifuged overFicoll-Hypaque and assessed as effector cells in a standard ⁵¹Cr-releaseassay using U-bottomed microtiter plates containing about 10⁴ targetcells with varying effector cell concentrations. All cells are assayedtwice. Autologous B lymphoblastoid cell lines are used as target cellsand are loaded with peptide by incubation overnight during ⁵¹Crlabeling. Specific release is calculated in the following manner:(experimental release-spontaneous release)/(maximum release-spontaneousrelease)×100. Spontaneous release is generally less than 20% of maximalrelease with detergent (2% Triton X-100) in all assays. A successfulCD8⁺ response occurs when the induced cytolytic activity is above 10% ofcontrols.

Another measure of CD8⁺ responses provides direct quantification ofantigen-specific T cells by staining with Fluorescein-labeled HLAtetrameric complexes (Altman, J. D. et al., Proc. Natl. Acad. Sci. USA90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996). Otherassays include staining for intracellular lymphokines, and γ-interferonrelease assays or ELISPOT assays. Tetramer staining, intracellularlymphokine staining and ELISPOT assays all are sensitive measures of Tcell response (Laivani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar,P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al.,Immunity 8:177, 1998).

Viral Titer

There are a variety of ways to measure viral titer in a patient. Areview of the state of this art can be found in the Report of the NIH ToDefine Principles of Therapy of HIV Infection as published in the;Morbidity and Mortality Weekly Reports, Apr. 24, 1998, Vol 47, No. RR-5,Revised Jun. 17, 1998. It is known that HIV replication rates ininfected persons can be accurately gauged by measurement of plasma HIVconcentrations.

HIV RNA in plasma is contained within circulating virus particles orvirions, with each virion containing two copies of HIV genomic RNA.Plasma HIV RNA concentrations can be quantified by either targetamplification methods (e.g., quantitative RT polymerase chain reaction[RT-PCR], Amplicor HIV Monitor assay, Roche Molecular Systems; ornucleic acid sequence-based amplification, [NASBA®], NucliSens™ HIV-1 QTassay, Organon Teknika) or signal amplification methods (e.g., branchedDNA [bDNA], Quantiplex™ HIV RNA bDNA assay, Chiron Diagnostics). ThebDNA signal amplification method amplifies the signal obtained from acaptured HIV RNA target by using sequential oligonucleotidehybridization steps, whereas the RT-PCR and NASBA® assays use enzymaticmethods to amplify the target HIV RNA into measurable amounts of nucleicacid product. Target HIV RNA sequences are quantitated by comparisonwith internal or external reference standards, depending upon the assayused.

Measurement of Antibody Response

The ability of a patient to mount a B-cell response to the chimericprotein can also be measured. Typically, a serum sample from the patientis assayed for the presence of antibodies after administration of thechimeric rev, tat, and nef polypeptide. Antibodies can be detected usinga variety of immunoassays, including competitive and non-competitiveformats. Such assays are described in a number of publications, e.g.,CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds., 1995-2001, JohnWiley & Sons, Inc.; Harlow & Lane, ANTIBODIES: A LABORATORY MANUAL, ColdSpring Harbor Laboratory Press, 1988; and Harlow & Lane, USINGANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press,1999.

Formulation of Vaccines and Administration

The administration procedure for recombinant virus or DNA is notcritical. Vaccine compositions (e.g., compositions containing thepoxvirus recombinants or DNA) can be formulated in accordance withstandard techniques well known to those skilled in the pharmaceuticalart. Such compositions can be administered in dosages and by techniqueswell known to those skilled in the medical arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the route of administration.

The vaccines can be administered prophylactically or therapeutically. Inprophylactic administration, the vaccines are administered in an amountsufficient to induce CD8⁺ and CD4⁺, or antibody, responses. Intherapeutic applications, the vaccines are administered to a patient inan amount sufficient to elicit a therapeutic effect, i.e., a CD8⁺, CD4⁺,and/or antibody response to the HIV-1 antigens or epitopes encoded bythe vaccines that cures or at least partially arrests or slows symptomsand/or complications of HIV infection. An amount adequate to accomplishthis is defined as “therapeutically effective dose.” Amounts effectivefor this use will depend on, e.g., the particular composition of thevaccine regimen administered, the manner of administration, the stageand severity of the disease, the general state of health of the patient,and the judgment of the prescribing physician.

The vaccine can be administered in any combination, the order is notcritical. In some instances, for example, a DNA HIV vaccine isadministered to a patient more than once followed by delivery of one ormore administrations of the recombinant pox virus vaccine. Therecombinant viruses are typically administered in an amount of about 10⁴to about 10⁹ pfu per inoculation; often about 10⁴ pfu to about 10⁶ pfu.Higher dosages such as about 10⁴ pfu to about 10¹⁰ pfu, e.g., about 10⁵pfu to about 10⁹ pfu, or about 10⁶ pfu to about 10⁸ pfu, can also beemployed. For example, a NYVAC-HIV vaccine can be inoculated by theintramuscular route at a dose of about 10⁸ pfu per inoculation, for apatient of 170 pounds.

Suitable quantities of DNA vaccine, e.g., plasmid or naked DNA can beabout 1 μg to about 100 mg, preferably 0.1 to 10 mg, but lower levelssuch as 0.1 to 2 mg or 1-10 μg can be employed. For example, an HIV DNAvaccine, e.g., naked DNA or polynucleotide in an aqueous carrier, can beinjected into tissue, e.g., intramuscularly or intradermally, in amountsof from 10 μl per site to about 1 ml per site. The concentration ofpolynucleotide in the formulation is from about 0.1 μg/ml to about 20mg/ml.

The vaccines may be delivered in a physiologically compatible solutionsuch as sterile PBS in a volume of, e.g., one ml. The vaccines can alsobe lyophilized prior to delivery. As well known to those in the art, thedose may be proportional to weight.

The compositions included in the vaccine regimen of the invention can beco-administered or sequentially administered with other immunological,antigenic or vaccine or therapeutic compositions, including an adjuvant,a chemical or biological agent given in combination with orrecombinantly fused to an antigen to enhance immunogenicity of theantigen. Additional therapeutic products can include biological responsemodifiers such as cytokines or co-stimulatory agents, e.g.,interleukin-2 (IL-2) or CD40 ligand in an amount that is sufficient tofurther potentiate the CD8⁺ and CD4⁺ T-cell responses.

Other compositions that can be administered with the vaccines of thecurrent invention include purified antigens from the immunodeficiencyvirus or proteins obtained from the expression of such antigens by asecond recombinant vector system. For example, additional compositionscan include vaccines, such as nucleic-acid based vaccines, that encodeother HIV proteins, for instance structural proteins, e.g., gag, pol,and env. Administration of these additional agents can occur before,after, or concurrently with adminsitration of the vaccines comprisingchimeric rev, tat, and nef.

Examples of adjuvants which also may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). Again, co-administration isperformed by taking into consideration such known factors as the age,sex, weight, and condition of the particular patient, and, the route ofadministration.

The peptide, viral and DNA vaccines can additionally be complexed withother components such as lipids, peptides, polypeptides andcarbohydrates for delivery. Such formulations are known in the art, see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publising Co., Easton, Pa., 1985)

The DNA vaccines are administered by methods well known in the art asdescribed in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997));Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner(U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.Pat. No. 5,679,647, issued Oct. 21, 1997). The vectors can also becomplexed to particles or beads that can be administered to anindividual, for example, using a vaccine gun. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the expression vector.

Vaccines may be delivered via a variety of routes. Typical deliveryroutes include parenteral administration, e.g., intradermal,intramuscular or subcutaneous delivery. Other routes include oraladministration, intranasal, and intravaginal routes. For DNA vaccines inparticular, the vaccines can be delivered to the interstitial spaces oftissues of an individual (Felgner et al., U.S. Pat. Nos. 5,580,859 and5,703,055). Administration of DNA vaccines to muscle is also afrequently used method of administration, as is intradermal andsubcutaneous injections and transdermal administration. Transdermaladministration, such as by iontophoresis, is also an effective method todeliver nucleic acid vaccines to muscle. Epidermal administration ofexpression vectors of the invention can also be employed. Epidermaladministration involves mechanically or chemically irritating theoutermost layer of epidermis to stimulate an immune response to theirritant (Carson et al., U.S. Pat. No. 5,679,647).

The vaccines can also be formulated for administration via the nasalpassages. Formulations suitable for nasal administration, wherein thecarrier is a solid, include a coarse powder having a particle size, forexample, in the range of about 10 to about 500 microns which isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable formulations wherein the carrier is aliquid for administration as, for example, nasal spray, nasal drops, orby aerosol administration by nebulizer, include aqueous or oilysolutions of the active ingredient. For further discussions of nasaladministration of AIDS-related vaccines, references are made to thefollowing patents, U.S. Pat. Nos. 5,846,978, 5,663,169, 5,578,597,5,502,060, 5,476,874, 5,413,999, 5,308,854, 5,192,668, and 5,187,074.

Examples of vaccine compositions of use for the invention include liquidpreparations, for orifice, e.g., oral, nasal, anal, vaginal, etc.administration, such as suspensions, syrups or elixirs; and,preparations for parenteral, subcutaneous, intradermal, intramuscular orintravenous administration (e.g., injectable administration) such assterile suspensions or emulsions. In such compositions the recombinantpoxvirus, expression product, immunogen, DNA, or modified gp120 or gp160may be in admixture with a suitable carrier, diluent, or excipient suchas sterile water, physiological saline, glucose or the like.

The vaccines can be incorporated, if desired, into liposomes,microspheres or other polymer matrices (Felgner et al., U.S. Pat. No.5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed.1993), each of which is incorporated herein by reference). Liposomes,for example, which consist of phospholipids or other lipids, arenontoxic, physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

Liposome carriers may serve to target a particular tissue or infectedcells, as well as increase the half-life of the vaccine. Liposomesinclude emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like. Inthese preparations the vaccine to be delivered is incorporated as partof a liposome, alone or in conjunction with a molecule which binds to,e.g., a receptor prevalent among lymphoid cells, such as monoclonalantibodies which bind to the CD45 antigen, or with other therapeutic orimmunogenic compositions. Thus, liposomes either filled or decoratedwith a desired immunogen of the invention can be directed to the site oflymphoid cells, where the liposomes then deliver the immunogen(s).Liposomes for use in the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome size,acid lability and stability of the liposomes in the blood stream. Avariety of methods are available for preparing liposomes, as describedin, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The dosage for an initial immunization generally occurs in a unit dosagerange where the lower value is about 1, 5, 50, 500, or 1,000 μg and thehigher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosagevalues for a human typically range from about 500 μg to about 50,000/gper 70 kilogram patient. Boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide pursuant to a boosting regimen over weeks tomonths may be administered depending upon the patient's response andcondition as determined by measuring the specific activity of CTL andHTL obtained from the patient's blood or by measuring antibody response.The dosages, routes of administration, and dose schedules are adjustedin accordance with methodologies known in the art.

The concentration of peptides of the invention in the pharmaceuticalformulations for administration as a vaccine can vary widely, i.e., fromless than about 0.1%, usually at or at least about 2% to as much as 20%to 50% or more by weight, and will be selected primarily by fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

A human unit dose form of the peptide composition is typically includedin a pharmaceutical composition that comprises a human unit dose of anacceptable carrier, preferably an aqueous carrier, and is administeredin a volume of fluid that is known by those of skill in the art to beused for administration of such compositions to humans (see, e.g.,Remington's Pharmaceutical Sciences, supra).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES Example 1 Preparation of an SIV retanef Chimeric Gene

The overall composition of the retanef gene is depicted on FIG. 1. Todisrupt the functional domains within the tat and nef genes, both geneswere split into two segments, and reassorted in the Retanef construct sothat the C-terminal part of Nef precedes its N-terminal part and the twoare separated by C-terminal part of Tat (FIG. 1). In order tominimize/prevent nuclear localization and RNA-binding of the Retanefprotein, the amino acids comprising the nuclear localization sequence(NLS) and RNA-binding domain of Rev (position 40-7) (Henderson et al., JMol Biol, 274:693-707 (1997); Truant et al., Mol Cell Biol, 19:1210-1217(1999)) and Tat (position 81-84) (Truant et al., Mol Cell Biol,19:1210-1217 (1999)) and Tat (position 81-84) (71) were deleted (FIG.2). The myristylation signal at the N-terminus of the Nef protein, anecessary requirement for translocation of Nef to cellular membrane anddownregulation of the CD4 and MHC-I molecules (Hua et al., Virology,231:231-238 (1997); Sawai et al., J Biol Chem, 270:15307-15314 (1995)),and key for viral pathogenicity in vivo (Aldrovandi et al., J Virol,72:7032-7039 (1998)), was deleted. In order to preserve putative CTLepitopes, the N- and C-terminus of Nef protein was designed with anoverlap of eight amino acids (HRILDIYL). The DNA sequence was modifiedby the use of different codons in the two overlapping regions tominimize the risk of recombination. The nef sequence was obtained fromthe SIVmac239 strain and the premature stop codon was mutagenagenizedfrom TAA to GAA. The known SIVmac239 Nef CTL epitopes recognized byinfected and vaccinated rhesus macaques (Allen et al., J. Virol.,75:738-749 (2001); Evans et al., J Virol, 74:7400-7410 (2000); Evans etal., Nat.Med., 5:1270-1276 (1999)) are conserved in the Retanefconstruct.

Because the codon usage of lentiviruses reduces the efficacy of proteintranslation in mammalian cells (Andre et al., J Virol, 72:1497-1503(1998); Haas et al., Curr Biol, 6:315-324 (1996)), the DNA sequence ofRetanef was designed using appropriate codons for expression inmammalian genes. The DNA sequences of the Tat and Rev were adapted fromknown SIVmac isolates. The HA.1 epitope tag was added at the C-terminusof the retanef gene to facilitate the detection of the chimeric protein.The RTN DNA was cloned into the pCMV/Kan plasmid under the control ofCMV promoter and into the highly attenuated poxvirus vector NYVAC underthe control of H6 promoter.

A description of the plasmid construction is provided below.

The sequences of rev and tat genes were derived from the publishedsequences of SIVmac251 isolates (Genbank accession numbers M19499,M15897, M16125, M24614, X06391, X06393, X06879, Y00283, Y00294, Y00295).The nef gene sequence was designed according to the sequence ofSIVmac239 nef with a TAA to GAA mutation in position E92 in order torepair the premature stop codon. The C-terminal part contains thesequence that repaired SIVmac239 nef shares with other SIVmac isolates(SIVML, Swiss-Prot #P11262; SIVM1, #P05862) and differs from thesequence of SIVMK isolate (SwissProt #P05861).

The Retanef construct also includes a sequence that reconstitutes the H6promoter in the pATIHIVMNT plasmid. Two restriction endonuclaserecognition sites for SpeI and SalI were inserted between C-Tat 2 andN-Nef in order to facilitate cloning of additional genes into retanef.An HA. 1 protein tag was attached to the C-terminus to facilitate thedetection of the protein using commercially available antibodies.

The retanef gene construct was synthesized by Midland Molecular BiologyGroup (Midland, Tex.). An SacII/EcORI fragment containing the Retanefconstruct was cloned into an expression vector derived from thekanamycin-expressing pVR1332 (Vical Inc.) (31) under the control of aCMV promoter. Plasmid preparations of clinical-grade quality wereproduced by Qiagen (Hilden, Germany). The NruI/XhoI Retanef fragment wascloned into pATIHIVMNT plasmid (Virogenetics, NY) and inserted byhomologous recombination into the NYVAC vector to obtain theNYVAC-SIV-rtn recombinant vaccine vp1658, as previously described(Benson et al., J. Virol., 72:4170-4182 (1998)).

Example 2 Expression and Cellular Localization of DNA-SIV-rtn and theRecombinant NYVAC-SIV-rtn in Monkey and Human Cells

Expression of the retanef gene was assessed by transfecting theDNA-SIV-rtn construct into Hela cells.

Transfection was performed as follows. Hela-Tat cells were plated at3×10⁵ cells/per 6 cm-diameter plates and after 16 hrs transfected by thecalcium phosphate method (28). For transfection, 1 μg of pCMV/Retanef orpCMV/Gag were used and the amount of transfected DNA was normalized to 2μg with pMEI8S expression vector obtained from Atsushi Miyajima (DNAX,Palo Alto, Calif.). Control cells were transfected with 2 μg of pME18SDNA. Twenty four hours later, the cells were lysed and the amount ofprotein was determined. Total cellular protein (30 μg) waselectrophoresed on a 10% SDS gel, transferred to a nitrocellulosemembrane, and analyzed by Western blotting using an anti-HA antibody,3F10-HRP (Roche Biochemicals, Indianapolis).

A 55 kDa protein was detected in the DNA-SIV-rtn-transfected cells butnot in the mock-transfected cells. Expression of the Retanef protein inthe cell lysate of African green monkey-derived Vero cells infected withNYVAC or the nonrecombinant NYVAC vector was also determined usingWestern blots. The 55 kDa Retanef protein was detected only in cellsinfected with NYVAC-SIV-rtn.

To determine the subcellular localization of the Retanef protein, Helacells were transfected with either DNA-SIV-rtn or control DNA-SIV-rtnGag plasmids and analyzed using an indirect immunofluorescence assaywhich was performed as described below.

Hela-Tat cells were seeded onto slides (2×10⁵ cells per slide) andtransfected the following morning. One-fifth of the above transfectionmix was used to transfect the cells on the slides. Twenty-four hourslater, cells were washed twice in PBS, fixed at room temperature for 7minutes in 2% paraformaldehyde in PBS, and incubated for 1 hour at 37°C. in 0.1% Saponin (Sigma). Slides were incubated with a 1:100 dilutionof anti-HA antibody (12CA5, Roche Biochemicals) in 10% FCS/PBS for 1hour at 37° C., washed four times in 0.02% Tween 20/PBS, and wereincubated as described for the primary antibody with a 1:1000 dilutionof a Cy2-conjugated anti-mouse antibody (Jackson ImmunOResearch, PA).After washing, cells were counterstained with Evan's Blue, washed, andstained with DAPI.

No specific staining was observed in cells transfected with either thecontrol plasmid or the pCMV Gag, whereas cells infected with theDNA-SIV-rtn cells were detected. The Retanef protein localized mainly inthe perinuclear and cytoplasmic compartments of the transfected cellsand did not appear to accumulate in the nucleus of the transfectedcells.

Example 3 DNA-SIV-rtn and NYVAC-SIV-rtn are Immunogenic in Naive RhesusMacaques

It has been shown that priming with DNA-SIV-gag-env (DNA-SIV-ge)followed by boosting with NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe) vaccinecandidate induces high frequency of virus specific CTLs and strong anddurable lymphoproliferative responses in rhesus macaques. In order toassess the immunogenicity of the DNA-SIV-rtn vaccine candidate, naiverhesus macaques were immunized with DNA-SIV-rtn by the intramuscular andintradermal routes three times and boosted with a single dose ofNYVAC-SIV-rtn at week 25. Immunization was performed as follows.

All animals were colony bred rhesus macaques (Macaca mulatta) obtainedfrom Covance Research Products (Alice, Tex.). Animals were housed andhandled in accordance with the standards of the Association for theAssessment and Accreditation of Laboratory Animal Care International(AAALAC International). All rhesus macaques were seronegative for SIV-1,STLV-1, and herpesvirus B prior to the study. Screening for the presenceof Mamu-A*01 allele was performed using PCR as described (Knapp et al.,Tissue Antigens, 50:657-661 (1997)). DNA immunization was performed asfollows: 4 mg of pCMV/Retanef or pCMV/Gag plasmid were administered in aregimen of 4 doses of 0.75 mg of each plasmid injected intramuscularlyinto 2 sites on each leg and 5 doses of 0.2 mg of each plasmid injectedintradermally at 5 different sites in the abdominal area. ForNYVAC-SIV-rtn immunization, animals were inoculated intramuscularly with10⁸ pfu of NYVAC-SIV-rtn per immunization.

Naive rhesus macaques 687, 688, and 820 were immunized with three dosesof DNA-SIV-rtn simultaneously by intramuscular and intradermal routes atweek 0, 4, and 12, followed by a boost with a single dose ofNYVAC-SIV-rtn vaccine given intramuscularly at week 25.

Epitope specific CD3+ CD8+ T lymphphocytes were detected by flowcytometry. Fresh PBMC were stained with anti-human CD3 Ab (PerCPlabeled, clone SP34, Pharmingen, San Diego, Calif.), anti-human CD8(FITC labeled, Becton-Dickinson, San Jose, Calif.), and Mamu-A*01tetrameric complexes refolded in the presence of a specific peptide(kindly provided by Dr. J. Altman) and conjugated to PE labeledstreptavidin (Molecular Probes, Eugene, Oreg.). Samples were analyzed ona FACSCalibur (Becton-Dickinson) and the data are presented aspercentage of tetramer positive cells of all CD3+ CD8+ lymphocytes.

Monkey IFN-γ specific ELISPOT kits manufactured by U-Cytech (Utrecht,The Netherlands) were used in order to detect the number of cellsproducing IFN-γ upon in vitro stimulation. Ninety-six well flat bottomplates were coated with anti-IFN-γ mAb MD-1 overnight at 4° C. andblocked with 2% BSA in PBS for 1 hour at 37° C. About 10⁵ cells per wellwere loaded in quadruplicates in RPMI-1640 containing 5% human serum and10 μg per ml of a specific peptide or 5 μg per ml Concanavalin A as apositive control. The plates were incubated overnight at 37° C., 5% CO₂,and developed according to the manufacturer's guidelines (U-Cytech).

CTL response was also measured using a CTL assay cytotoxicity assay.About 5×10⁶ PBMC were cultured with 10 μg/ml of specific peptide forthree days, IL-2 (Roche, Indianopolis, Ind.) was added at 40 IU/ml andthe cells were cultured for another four days. Twelve hours before thekilling assay a second dose of IL-2 at 40 IU/ml was added. The cellswere then incubated for 6 hours in various effector to target cellratios with Mamu-A*01-positive ⁵¹Cr-labeled transformed B cells pulsedovernight with 10 μg per ml of a specific peptide. The killing of cellspulsed with an unrelated peptide in a control experiment was equal tothe killing observed in the absence of any peptide.

The analysis of vaccine-induced immune responses showed that two weeksfollowing the last immunization, a response to the immunodominantMamu-A*01-restricted CTL Tat epitope TTPESANL (Tat28) (4) was detectedin both Mamu-A*01 positive animals (687 and 688) by staining with aTat28-specific tetramer at two weeks following the boost. Cells from theMamu-A*01-negative control macaque 820 did not stain with the tetramer,demonstrating the specificity and MHC-I restriction of this tetramermolecule.

The functionality of these Tat-specific responses was assayed usingIFN-γ ELISPOT. An increase in the number of cells producing IFN-γfollowing in vitro stimulation with the Tat28 peptide was observed afterone week from the last immunization. Furthermore, the cells from bothMamu-A*01-positive vaccinated animals, but not from twoMamu-A*01-positive naive control animals, were able to lyse Tat28peptide-pulsed ⁵¹Cr-labeled target B-cells following a 7-day expansionin culture in the presence of the Tat28 peptide.

Thus, the use of three different assays demonstrated the immunogenicityof the Retanef-based vaccine in naive rhesus macaques.

CD4⁺ T-cell proliferative responses to Tat and Nef were also assessed atlater time points. Both Tat-specific and Nef-specific T-cellproliferation was observed over 52 weeks after initiation of the vaccineregimen (FIGS. 3 and 4, top panels).

Induction of immune responses to individual antigens in animalsvaccinated with DNA/NYVAC-SIV-retanef alone was compared to thosevaccinated with both DNA/NYVAC-SIV-retanef andDNA/NYVAC-SIV-gag-pol-env. Animals were vaccinated withDNA/NYVAC-SIV-gag-pol-env (Hel et al., J Immunol 67:7180-91, 2001). Theretanef and gag-pol-env DNA constructs were administered at the sametime, but at different sites using the DNA/NYVAC regimen previouslydescribed (Hel et al., J Immunol 67:7180-91, 2001). The time frame anddose of of administration, i.e., t Tat-specific and Nef-specific CD4+proliferation was generally reduced in comparison to those animals thatwere inoculated only with DNA/NYVAC-SIV-retanef (FIGS. 3 and 4, lowerpanels). Furthermore, tetramer staining with Tat 28 showed that inmacaques that were immunized only with the Retanef construct, by day 17,the Tat response was more than 4%, whereas it reached the same level inboth animals vaccinated with all antigens by week 3 (FIG. 5). Despite adecrease of the relative amounts of the virus-specific immune response,a significantly lower degree of viremia during acute infection wasobserved in the primary viremia of macaques immunized with all antigens(group E, FIGS. 6 and 7). These data suggest that vaccination with allof the antigens improved the virological outcome during primaryinfection and moreover, suggest that sequential administration of retanfwith other vaccines, e.g., those inducing responses to the structuralproteins, gag, pol, and env can be preferable.

Example 4 Adminstratin of DNA-SIV-rtn and NYVAC-SIV-rtn to SIV-InfectedAnimals

Active immunization of HIV-1-infected HAART-treated individuals maycontribute to transient viremia containment in the absence of HAART(Gotch et al., Immunol Rev, 170:173-182 (1999); Hel et al., Nat.Med.,6:1140-1146 (2000)). The immunogenicity of both DNA-SIV-rtn andNYVAC-SIV-rtn vaccine candidates in chronically SIVmac251 infectedmacaques treated with antiretroviral therapy (ART) was thereforeassessed.

Macaques 454, 455, 460, and 541 were infected for 16 months following anintrarectal challenge exposure to a pathogenic SIVmac251 and subjectedto antiretroviral therapy for 14 weeks prior to an inoculation with asingle dose of either DNA-SIV-rtn (macaques 454 and 455) or DNA-ge(macaques 460 and 541).

Macaques 3075, 3057, and 3077 were exposued intravenously to SIVmac251virus and became infected and were started on ART 6 months afterinfection. The macaques were treated with ART for 8 months prior to asingle innoculation of NYVAC-SIV-rtn vaccine candidate (macaques 3075and 3057) or control mock NYVAC (macaque 3077). Antiretroviral therapyconsisted of subcutaneous inoculation of 20 mg/kg/day of PMPA[(R)-9-(2-phosphonylmethoypropyl)adenine], oral administration of 2.4mg/kg/day of Stavudine (d4T) divided into 2 doses daily, and intravenousinoculation of 10 mg/kg/day of Didanosine (DDI) as described previously(Hel et al., Nat.Med., 6:1140-1146 (2000)).

Both macaques inoculated with a single dose of DNA-Retanef givenintramuscularly and intradermally (macaques 454 and 455), but none ofthe macaques immunized with DNA-gag-env (DNA-ge) (macaques 460 and 541)exhibited a three- to sevenfold increase in the frequency of Tat28tetramer-staining cells in blood over a two weeks period following thevaccination. Likewise, both DNA-ge immunized macaques, but not theDNA-SIV-rtn immunized animals had an increased number of cells stainingwith a tetramer specific for a Gag epitope Gag181 after immunization(data not shown), demonstrating once again the specificity of stainingwith the Tat28 tetramer. A three- to fivefold increase in the frequencyof Tat28-specific cells was measured in the blood of both theNYVAC-SIV-rtn immunized macaques. Thus, both the DNA-SIV-rtn andNYVAC-SIV-rtn vaccine candidates increased the SIV Tat specific CD8+T-cell response by several fold in SIVmac251 chronically infectedART-treated animals.

The above examples describe the design, immunogenicity, and safety ofnovel vaccine candidates expressing retroviral early regulatory genes inboth naive and chronically infected macaques treated with antiretroviraltherapy. These vaccine candidates were designed with the aims tominimize possible negative affects of Tat and Nef and to ameliorateexpression of these antigens in primate cells. Chimeric Retanef wasefficiently expressed in both human and monkey cells and did notaccumulate in the nucleus of the transfected cells. Furthermore, boththe DNA-SIV-rtn and NYVAC-SIV-rtn vaccine candidates were able to expanda virus-specific CD8+ T-cell response to Tat, as measured by directtetramer staining in the blood of macaques naive or chronically infectedwith SIVmac251 and treated with antiretroviral therapy. In naivemacaques, the increase of frequency in the Tat response was alsoconfirmed using functional assays such as IFN-γ production and cytolyticactivity.

The relative immunogenicity of Tat, Rev, and Nef expressed using a DNAplasmid or within a recombinant NYVAC vector can be compared to theknown immunogenicity of the individual antigens. For example, theimmunogenicity of biologically active Tat versus a Tat toxoid did notappear to differ significantly, and in neither case was notableprotection from infection or high viremia was observed followingchallenge exposure (Pauza et al., Proc. Natl. Acad. Sci. USA,97:3515-3519 (2000); Rappaport et al., J Leukoc Biol, 65:458-465 (1999))(J. Shiver, personal communication). This suggests that the use of Tatas a single component of a vaccine for HIV-1 may not be a viableapproach.

Example 5 Administration of a Chimeric rev-tat-nef Vaccine to Prevent orTreat HIV Infection

An example of use of the vaccine to treat a patient at risk for HIV-1infection is provided in this Example. A patient is injected with anattenuated pox virus vector NYVAC carrying a ref-tat-nef chimeric genedesigned in accordance with Example 1. The injection comprises about 10⁸pfU of the pox virus.

The patient's immune responses is evaluated (CD4+ proliferativeresponse; cytotoxic CD8+ T-cell activity, etc.) and a decision is madeas to whether and when to immunize again. Typically, a maximum of threeto four immunizations with NYVAC-ref-tat-nef is considered. This regimencould be followed by three to four immunizations with ALVAC-ref-tat-nef(carrying a similar HIV-I genetic content). A DNA-only i.e., DNA that isnot in a viral vector, vaccine can also be administered, e.g., precedingNYVAC administration.

The vaccine regimen is administered with IL-2, preferably at low dosessuch as 100,000 to 200,000 units of IL-2 administered daily. CD40⁺ligand can also be included in the treatment protocol, either by itselfor administerd in conjunction with the IL-2 treatment.

The examples provided are by way of illustration only and not limiting.Those of skill will readily recognize a variety of noncriticalparameters which could be changed or modified to yield essentiallysimilar results.

1. An expression vector comprising a nucleic acid encoding a chimeric rev, tat, and nef polypeptide.
 2. An expression vector of claim 1, wherein the vector is a viral vector.
 3. The expression vector of claim 2, wherein the viral vector is an attenuated pox virus vector.
 4. The expression vector of claim 3, wherein the attenuated pox virus vector is NYVAC, ALVAC, MVA, or fowlpox.
 5. The expression vector of claim 1, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, or a Venezuelan equine encephalomyelitis virus vector.
 6. The expression vector of claim 1, wherein the chimeric polypeptide expressed by the vector comprises functional domains of tat and nef that are disrupted.
 7. The expression vector of claim 1, wherein the chimeric polypeptide expressed by the vector lacks a Rev nuclear localization signal, a Rev RNA-binding domain, and a Tat RNA-binding domain.
 8. The expression vector of claim 1, wherein the chimeric polypeptide expressed by the vector lacks an N-terminal Nef myristylation signal.
 9. The expression vector of claim 1, wherein the chimeric polypeptide expressed by the vector is Retanef.
 10. The expression vector of claim 9, further wherein the expression vector is an attenuated pox virus vector that is NYVAC or ALVAC.
 11. A method of inducing an immune response comprising administering a first expression vector comprising a nucleic acid sequence encoding a chimeric rev, tat, and nef polypeptide, wherein the expression vector enters the cells of the recipient and intracellularly produce rev, tat, and nef-specific peptides that are presented on the cell's MHC class I molecules in an amount sufficient to stimulate a CD8⁺ response.
 12. The method of claim 11, wherein the chimeric rev, tat, and nef polypeptide is retanef.
 13. The method of claim 11, wherein the expression vector is a viral vector.
 14. The method of claim 13, wherein the viral vector is an attenuated pox virus vector.
 15. The method of claim 14, wherein the attenuated pox virus vector is selected from the group consisting of NYVAC and ALVAC.
 16. The method of claim 13, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, or a Venezuelan equine encephalomyelitis virus vector.
 17. The method of claim 12, further wherein the chimeric rev, tat, and nef polypeptide is retanef.
 18. The method of claim 11, further comprising administering a second expression vector comprising a nucleic acid sequence encoding a chimeric rev, tat, and nef polypeptide, wherein the second expression vector is administered as naked DNA.
 19. The method of claim 18, further comprising administering a third expression vector comprising a nucleic acid sequence encoding an HIV structural protein.
 20. The method of claim 19, wherein the expression vector encodes HIV gag, pol, and env.
 21. The method of claim 18, wherein the chimeric rev, tat and nef polypeptide is retanef.
 22. A chimeric rev, tat, and nef polypeptide.
 23. The chimeric polypeptide of claim 22, wherein the polypeptide is Retanef.
 24. A method of inducing an immune response comprising administering a peptide of claim
 22. 